Chemotherapy-induced primary ovarian insufficiency is an imminent health concern. Progressing female cancer survivorship demands new approaches to prevent unintended chemotherapy-induced primary ovarian insufficiency (POI) and subsequent early menopause. Premature menopause increases a woman's risk for complications due to estrogen depletion including osteoporosis, mental health disorders, and cardiovascular disease. By the year 2020, it is estimated that 1 in 500 adult women will be survivors of childhood cancer, over 8% of whom will experience POI.
The only available prophylactic fertility preservation therapy for prepubertal cancer patients who become cancer survivors requires surgically removing and freezing ovarian tissue prior to gonadotoxic cancer treatment for future re-transplantation. While the transplanted tissue provides fertility and natural hormonal cycling for a limited time, the procedure is expensive, invasive, considered experimental, and carries the risk of reintroducing the original cancer [1-5].
POI occurs more frequently in adult cancer patients, with up to 40% of breast cancer survivors suffering from the disorder [6-8]. Fertility preservation options for reproductively-mature female cancer patients include oocyte and embryo cryopreservation, but the requisite hormone treatments are contraindicated for patients with estrogen-responsive tumors, may delay cancer treatment, and do not preserve endocrine function. Oocyte and embryo cryopreservation are expensive procedures, have a modest success rate, expose the patient to considerable medical risks, and do not preserve overall endocrine health (normal hormonal regulation) nor prevent premature menopause.
Doxorubicin (DXR) Ovarian Toxicity.
DXR is an anthracyline used to treat roughly 50% of all cancer cases [9-11], and is associated with POI. Cancer therapies that typically utilize DXR generally have higher survival rates than others, but there are no clinical therapeutics to prevent DXR-induced POI; it is therefore these future survivors who will benefit from the drug-based ovarian shield proposed here. DXR can cause cell death via two distinct mechanisms; (1) intercalating into DNA and thus preventing resealing of topoisomerase II (topoII)-dependent double-strand DNA (dsDNA) breaks, and (2) inducing oxidative stress [12-18]. TopoII-dependent dsDNA damage appears to be the mechanism of DXR insult in ovarian cells [19] and requires drug transport into the cell nucleus where intercalation occurs.
Previous studies have shown DXR treatment induces a bi-temporal response in the mouse ovary with follicular apoptosis by 12 hours (hrs) post-injection [20, 21], followed by a return to 50% pre-DXR ovulation rate at 1 month post-DXR [21], and long-term follicular depletion [22, 23]. Oocytes exposed to DXR in vitro can undergo oxidative stress [20, 24-29] and exhibit chromosome condensation [30, 31].
To generate an in vivo model for testing putative ovoprotective agents, we previously characterized temporal and spatial accumulation of DXR in the mouse ovary, DXR-induced DNA damage, and consequent apoptosis [32]. These data demonstrate that DXR insult in the ovary is complex, involving responses that are cell- and follicle-type specific. DXR accumulates first in stromal cells, quantifiable by confocal microscopy at 2 hrs post-injection, then continuously shifts distribution to accumulate in follicles. Direct DXR-induced DNA damage prior to apoptosis was quantified using the neutral comet assay (NCA) in ovarian cells isolated from DXR-treated mice. This sensitive, single-cell electrophoretic assay reveals DNA damage in stromal/theca cells earlier than granulosa cells (2 hrs vs. 4 hrs post-injection, respectively).
As the first site of DXR-induced DNA damage, protecting stromal cells from chemotherapy insult may be critical to shielding the ovary as a whole. Stromal cells provide structural support for the ovary and determine the extracellular matrix composition, which in turn influences follicular maturation. Granulosa cells appear more sensitive to DXR-induced DNA damage, however, with an approximate 2-fold increase over control compared to a maximal 50% increase in stromal cells. It is therefore equally important to shield the granulosa cells, which maintain follicular health and nourish the oocyte, from chemotherapy.
Oocytes did not exhibit a significant increase in DNA damage over control until 10-12 hrs post-injection, a comparatively late sequel to granulosa cell damage occurring only after significant TUNEL signal in the granulosa cells indicates late-stage apoptosis and failing follicles. By 8 hrs post-DXR, antral follicles exhibit a 100% apoptotic index, and by 12 hours, secondary follicles plateau at 40% and primary follicles reach a 12% apoptotic index. These data suggest oocytes are either late targets of DXR or fail subsequent to follicular deterioration. Apoptotic events in primordial follicles (PFs) are not detected until 48 hrs post-DXR, despite significant DXR accumulation. The PFs are the follicle population which constitute the ovarian reserve and thus determine long-term fertility. PFs do sustain DXR-induced DNA damage, —as indicated by the appearance of phosphorylated γH2AX foci, the earliest cellular marker of dsDNA breaks, 48 hrs post-DXR. The complex ovarian response to DXR indicates that a successful ovarian protective agent needs to protect each ovarian cell type, as well as follicles at multiple stages.
Proteasome Inhibitors as Putative Ovoprotective Agents.
Though permeant to the cell plasma membrane, DXR is co-translocated across the nuclear membrane with the proteasome [33, 34], providing a potential mechanism to intercept nuclear DXR accumulation. The proteasome itself is responsible for over 90% of cellular protein turnover [35]. To regulate nuclear protein turnover, the assembled, active proteasome complex is translocated from the cytosol to the nucleus based in part on nuclear localization signals [36]. The proteasome is not structured like traditional transporters nor is the physiologic function drug transport, but the proteasome does mediate DXR nuclear accumulation [33, 37, 38]. Inhibitors including MG-132 and bortezomib (Bort), an aldehyde and boronate peptide, respectively, bind the proteasome active site with high affinity and specificity. Both MG-132 and Bort prevent DXR nuclear accumulation in L1210 cells by competing with DXR for binding to the proteasome active site [33]. MG-132 also prevents DXR-induced DNA damage in cardiac-derived H9C2 cells [39].
Well-tolerated in normal tissue, Bort is already approved for clinical use in anti-cancer therapies. Bort sensitizes myelomas and lung cancers to traditional chemotherapy, and is being tested to treat a variety of other cancers [40-107]. The toxicity in cancer cells is due to their requirement for rapid NF-κB turnover mediated by the proteasome to facilitate DNA transcription and rapid cell division [108-110].
Proteasome Inhibitor-mediated Ovoprotective Shielding Across Chemotherapy Drug Classes.
A further challenge facing the field of oncofertility is to avoid a scenario in which patients require an ovoprotective agent to correspond to each drug in their chemotherapy cocktail. The first member of the platinum-containing anti-cancer drugs, cisplatin is another common chemotherapy agent associated with high risk for POI [111]. Platinum drugs bind DNA and induce crosslinking which ultimately leads to apoptosis. Cisplatin is used in combination with DXR to treat hepatoblastoma (childhood liver cancer), neuroblastomas, osteosarcomas, Ewing and soft tissue sarcomas, endometrial cancer, and some triple negative breast cancers. In the rodent ovary, a single dose of cisplatin causes primordial follicle and oocyte destruction, decreases pregnancy rates and pups per litter in mice, and decreases circulating and follicular levels of anti-mullerian hormone (AMH) in rats [112-116]. Circulating AMH levels correlate with ovarian reserve such that a decrease in AMH is an indicator of POI. Also toxic to other organs, cisplatin causes nephrotoxicity by inducing depletion of the antiapoptotic protein, Mcl-1, and subsequent mitochondrial release of AIF [117]. Bort shields the kidney from cisplatin toxicity by preserving Mcl-1 levels [117]. Mcl-1 plays a critical role in follicle turnover as well [118-122]. This suggests Bort may also effectively shield the ovary from the platinum drug, albeit via a different mechanism than DXR shielding: preventing cisplatin-induced Mcl-1 depletion.
There is a critical need to develop a drug-based ovarian shield given routinely at the time of chemotherapy treatment to preserve both fertility and ovarian estrogen in female cancer patients regardless of age and cancer type. The long-term health consequences of early menopause in cancer survivors are expensive, and can include fertility treatment (IVF), as well as life-long treatment for osteoporosis, heart disease, and mental disorders as a result of estrogen depletion. Drug-based chemoprotection has the potential to overcome current obstacles in oncofertility by preserving ovarian endocrine function in a cost-effective, easily administered, non-invasive manner and avoiding health complications associated with premature menopause.